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Journal of Chromatography, 513 (1990) 4014Elsevier Science Publishers B.V., AmsterdamCHROM. 22 451

Note

Use of thin-layer chromatography for the testing ofavermectins produced by Streptomyces avermitdis strainsM. MALANfKOVA and V. MALANfKResearch I nstitute for F eed Supplements and Veteri nary Drugs, 281 61 Koui im (Czechoslovakia)andM. MAREK*Biotechnology Centre, Department of Biochemistry and M icr obiology, I nstitute of Chemical Technology, 16628 Prague (Czechoslovakia)(First received November 7th, 1989; revised manuscript received February 27th, 1990)

Avermectins are macrocyclic disaccharide antiparasitic agents having anthel-mintic and insecticidal properties and are produced by fermentation of Streptomycesavermitilis. Their structure was described2 as sixteen-membered lactones containingan a-L-oleandrosyl-a-L-oleandrosyl disaccharide attached to the lactone ring throughthe allylic Cr 3 hydroxy group. The complex contains four closely related major com-ponents, Ala, A2a, Bra and B2a, in varying proportions and four minor components,Arb, A2b, Bib and B2b, each of which is a lower homologue of the correspondingmajor component.

Avermectins do not show any antibiotic activity and therefore instead of micro-biological methods for their determination a biological test based on the movementparalysis of the free-living nematode Caenorhabditis elegans is commonly used3*4.The test is simple, but for a higher accuracy of determination of individual majorcomponents in an avermectin complex it is desirable to combine it with a chroma-tographic assay. The avermectin complex produced by investigated microorganismstrains can be tested by means of thin-layer chromatography (TLC) of extracts of thecultivating medium. The advantages of this technique are its simplicity and rapidityand the possibility of simultaneously determining a larger number of samples.However, the separation of the major compounds under the TLC conditionsdescribed so far13295-7 s not sufficient to make a densitometric determination pos-sible. The purpose of this investigation was to achieve an effective TLC separation ofthe major components Ala, Aza, Bra and B2a of avermectins, permitting their densi-tometric determination.EXPERIMENTALPreparation of samplesThe samples for TLC were prepared by extracting (5 min) the homogeneous0021-9673/90/$03.50 0 1990 Elsevier Science Publishers B.V.

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402 NOTES

Fig. 1. TLC of an extract from the cultivation medium of Streptomyces avermirilis producing avermectins.Solvent system: hexane-acetone-methanol(14:6:0.05). R, values of avermectins: A, = 0.41; A, = 0.36; B,= 0.26; B, = 0.20. Detection at 254 nm.

cultivation medium (5 ml) with a volume of chloroform so as to give a final avermec-tin concentration in the organic layer of cu. 1 mg/ml. The chloroform layer wasseparated by centrifugation (10 min, 5000 g).Thin-layer chromatographyThe samples (5-10 ~1 of organic layer) were applied to TLC plates (LK6F, 20 X20 cm; Whatman, Maidstone, U.K.) with a Linomat III instrument (Camag, Mut-tenz, Switzerland). The plates were developed for 30-40 min with different solventsystems. The best results were obtained with hexane-acetone-methanol(14:6:0.05) orhexane-isopropyl alcohol-methanol (14:4:0.05). For quantitative evaluation, theplates were measured at 254 nm by means of a TLC Scanner II densitometer (Camag)with Hewlett-Packard HP 3390 A integrator. Typical chromatograms are shown inFigs. 1 and 2. The concentrations of individual types of avermectin were determinedfrom a calibration graph (Fig. 3). Ivermectin [22,23-dihydrogen avermectins (Merk,St. Louis, MO, U.S.A.) (modified B1a and Bib avermectins giving one peak only onTLC plates), similarly to a main component Ai which is a mixture of Ala and Albcompounds; see below] was used as a standard.

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NOTES

0 0.2 0.6Fig. 2. TLC of an extract from the cultivation medium of Sfreptomyces avermitih producing avermectins.Solvent system: hexan+isopropyl alcohol-methanol (14:4:0.05). R, values of avermectins: A, = 0.53; A,= 0.45; B, = 0.38; B, = 0.30. Detection at 254 nm.

12 lb 20~ofhJumutin (PI

Fig. 3. Dependence of the peak area (measured densitometrically on TLC plates) on the amount ofivermectin.

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404 NOTESRESULTS

Different solvent systems were tested for the determination of individual aver-mectins in cultivation media by TLC: chloroform-ethyl acetate-methanol-methylenechloride (9:9: 1:2) (according to ref. l), ethyl acetate-ethanol (95:5), methylene chlo-ride-methanol (95:5 and 98:2), diethyl ether-light petroleum (30:70 and 180:20), hex-ane-ethyl acetate (85:5) (according to ref. 6), hexane-ethyl acetate (2:1), methylenechloridetetrahydrofuran-ethanol (90:9.7:0.3 and 89.7:10:0.3) (according to ref. 7),and some other combinations of these solvents. The best results were obtained withthe systems hexane-acetone-methanol (14:6:0.05) and hexane-isopropyl alcohol-methanol (14:4:0.05). Under these conditions, the mixture of avermectins was sep-arated into four main components, AI, AZ, B1 and Bz, each containing both themajor and the minor parts (e.g., component AI included compounds designated Alaand Arb). From a comparison of Figs. 1 and 2 it is evident that the second solventsystem gives a better separation of the components, with higher RF values. The deter-mination of individual avermectins in the cultivation medium based on measurementof corresponding peak areas using the calibration graph (Fig. 3) showed an inter-assay deviation of f 10% under standard conditions. The shapes of the peaks of theindividual avermectins and the ivermectin standard were very similar over the wholesample concentration range applied (the percentage deviation may be affected bypossible differences in the molar absorptivities for the individual avermectins andivermectin).The TLC technique described here permits an effective screening of Strepto-myces avermitilis strains from the viewpoint of the qualitative and quantitative com-position of the avermectins produced.REFERENCES1 R. W. Burg, B. M. Miller, E. E. Baker, J. Birnbaum, S. A. Currie, R. Hartman, Y.-L. Kong, R. L.Monaghan, G. Olson, J. Putter, J. B. Tunac, H. Wallick, E. 0. Stapley, R. Giva and S. Gmura, Anti-microb. Agents Chemother., 15 (1979) 361-367.

2 G. Alberts-Schdnberg, B. H. Arison, J. C. Chabala, A. W. Douglas, P. Eskola, M. H. Fischer, A. Lusi,H. Mrozik, J. L. Smith and R. L. Tolman, J. Am. Chem. Sot., 103 (1981) 4216-4221.3 S. Brenner, Genetics, 77 (1974) 71-94.4 J. M. Schaeffer and H. W. Haines, Biocbem. Pharmacol., 38 (1989) 2329-2338.5 M. H. Fisher, A. Lusi and R. L. Tolman, U.S. Put., 4 200 581 (1980).6 H. Mrozik, B. 0. Linn, P. Escola, A. Lusi, A. Matzuk, F. A. Preiser, D. A. Ostlind, J. M. Schaeffer andM. H. Fisher, .I. Med. Chem., 32 (1982) 375-384.7 H. Mrozik, P. Eskola, B. H. Arison, G. A. Schiinberg and M. H. Fisher, J. Org. Chem., 47 (1982)489492.